Compensator for multiple surface imaging

ABSTRACT

A system and method for imaging biological samples on multiple surfaces of a support structure are disclosed. The support structure may be a flow cell through which a reagent fluid is allowed to flow and interact with the biological samples. Excitation radiation from at least one radiation source may be used to excite the biological samples on multiple surfaces. In this manner, fluorescent emission radiation may be generated from the biological samples and subsequently captured and detected by detection optics and at least one detector. The detected fluorescent emission radiation may then be used to generate image data. This imaging of multiple surfaces may be accomplished either sequentially or simultaneously. In addition, the techniques of the present invention may be used with any type of imaging system. For instance, both epifluorescent and total internal reflection methods may benefit from the techniques of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

ThisMore than one reissue application of U.S. Pat. No. 9,365,898 hasbeen filed. A continuation reissue of the instant application,application Ser. No. 16/000,720, has been filed on Jun. 5, 2018. Theinstant application is a reissue application of U.S. Pat. No. 9,365,898,which issued on Jun. 14, 2016 from U.S. patent application Ser. No.14/721,870, which is herein incorporated in its entirety by reference,which is a continuation of U.S. patent application Ser. No. 14/229,455,entitled “Compensator for Multiple Surface Imaging,” filed Mar. 28,2014, and issued as U.S. Pat. No. 9,068,220 on Jun. 30, 2015, which isherein incorporated in its entirety by reference, and which is acontinuation of U.S. patent application Ser. No. 14/056,590, entitled“Compensator for Multiple Surface Imaging,” filed Oct. 17, 2013, andissued as U.S. Pat. No. 8,698,102 on Apr. 15, 2014, which is hereinincorporated in its entirety by reference, and which is a continuationof U.S. patent application Ser. No. 13/974,976, entitled “Compensatorfor Multiple Surface Imaging,” filed Aug. 23, 2013, and issued as U.S.Pat. No. 8,586,947 on Nov. 19, 2013, which is herein incorporated in itsentirety by reference, and which is a continuation of U.S. patentapplication Ser. No. 13/629,949, entitled “Compensator for MultipleSurface Imaging,” filed Sep. 28, 2012, and issued as U.S. Pat. No.8,546,772 on Oct. 1, 2013, which is herein incorporated in its entiretyby reference, and which is a continuation of U.S. patent applicationSer. No. 13/544,716, entitled “Compensator for Multiple SurfaceImaging,” filed Jul. 9, 2012, and issued as U.S. Pat. No. 8,278,630 onOct. 2, 2012, which is herein incorporated in its entirety by reference,and which is a continuation of U.S. patent application Ser. No.13/399,820, entitled “Compensator for Multiple Surface Imaging,” filedFeb. 17, 2012, and issued as U.S. Pat. No. 8,242,463 on Aug. 14, 2012,which is herein incorporated in its entirety by reference, and which isa continuation of U.S. patent application Ser. No. 13/281,237, entitled“Compensator for Multiple Surface Imaging,” filed Oct. 25, 2011, andissued as U.S. Pat. No. 8,143,599 on Mar. 27, 2012, which is hereinincorporated in its entirety by reference, and which is a continuationof U.S. patent application Ser. No. 13/209,306, entitled “Compensatorfor Multiple Surface Imaging,” filed Aug. 12, 2011, and issued as U.S.Pat. No. 8,071,962 on Dec. 6, 2011, which is herein incorporated in itsentirety by reference, and which is a continuation of U.S. patentapplication Ser. No. 12/434,495, entitled “Compensator for MultipleSurface Imaging,” filed May 1, 2009, and issued as U.S. Pat. No.8,039,817 on Oct. 18, 2011, which is herein incorporated in its entiretyby reference, and which claims priority of U.S. Provisional patentapplication Ser. No. 61/050,522, entitled “Multi-Surface BiologicalSample Imaging System and Method,” filed May 5, 2008, which is hereinincorporated in its entirety by reference, and of U.S. Provisionalpatent application Ser. No. 61/138,444, entitled “Compensator forMultiple Surface Imaging,” filed Dec. 17, 2008, which is hereinincorporated in its entirety by reference.

BACKGROUND

The present invention relates generally to the field of imaging andevaluating analytical samples. More particularly, the invention relatesto a technique for imaging and evaluating analytical samples on multiplesurfaces of a support structure using a compensator.

There are an increasing number of applications for imaging of analyticalsamples on a support structure. These support structures may includeplates upon which biological samples are present. For instance, theseplates may include deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) probes that are specific for nucleotide sequences present in genesin humans and other organisms. Individual DNA or RNA probes can beattached at specific locations in a small geometric grid or array on thesupport structure. Depending upon the technology employed, the samplesmay attach at random, semi-random or predetermined locations on thesupport structure. A test sample, such as from a known person ororganism, can be exposed to the array or grid, such that complementarygenes or fragments hybridize to probes at the individual sites on asurface of a plate. In certain applications, such as sequencing,templates or fragments of genetic material may be located at the sites,and nucleotides or other molecules may be caused to hybridize to thetemplates to determine the nature or sequence of the templates. Thesites can then be examined by scanning specific frequencies of lightover the sites to identify which genes or fragments in the sample werepresent, by fluorescence of the sites at which genes or fragmentshybridized.

These plates are sometimes referred to as microarrays, gene or genomechips, DNA chips, gene arrays, and so forth, and may be used forexpression profiling, monitoring expression levels, genotyping,sequencing, and so forth. For example, diagnostic uses may includeevaluation of a particular patient's genetic makeup to determine whethera disease state is present or whether pre-disposition for a particularcondition exists. The reading and evaluation of such plates are animportant aspect of their utility. Although microarrays allow separatebiological components to be presented for bulk processing and individualdetection, the number of components that can be detected in a singleexperiment is limited by the resolution of the system. Furthermore, thebulk reagents used in some methods can be expensive such that reducedvolumes are desired. The present invention provides methods andcompositions that increase the efficiency of array based detection tocounteract these limitations. Other advantages are provided as well andwill be apparent from the description below.

BRIEF DESCRIPTION

The present invention provides a novel approach to analytical sampleimaging and evaluation that expands the use of imaging and evaluationsubsystems to multiple surfaces that support samples. The supportstructure may, for instance, be a flow cell through which a reagentfluid is allowed to flow and interact with biological samples.Excitation radiation from at least one radiation source may be used toexcite the biological samples on multiple surfaces. In this manner,fluorescent radiation may be emitted from the biological samples andsubsequently captured and detected by detection optics and at least onedetector. The returned radiation may then be used to generate imagedata. This imaging of multiple surfaces may be accomplished eithersequentially or simultaneously. In addition, the techniques of thepresent invention may be used with any of a variety of types of imagingsystems. For instance, both epifluorescent and total internal reflection(TIR) methods may benefit from the techniques of the present invention.In addition, the biological samples imaged may be present on thesurfaces of the support structure in random locations or in patterns.

Accordingly, the invention provides a method for imaging a biologicalsample. The method includes detecting radiation emitted from a firstemissive component of a biological sample disposed on a first surface ofa flow cell using a detector. The flow cell is mounted on an imagingstation. The method also includes inserting corrective optics betweenthe detector and the flow cell. The method further includes detectingradiation emitted from a second emissive component of a biologicalsample disposed on a second surface of the flow cell using the detectorand the corrective optics. The first and second surfaces are in anarrangement whereby one of the surfaces is disposed between the detectorand the other surface. In addition, the corrective optics reduceaberration of detection at one of the surfaces due to the arrangement.The steps of the method are repeated while maintaining the flow cell onthe imaging station.

The invention further provides an imaging system for detecting radiationon a multi-surface flow cell. The imaging system includes amulti-surface flow cell having first and second emissive components of abiological sample disposed on respective first and second surfaces ofthe flow cell. The imaging system also includes an optical trainincluding an objective, imaging optics configured to focus the opticaltrain on the first emissive component via the objective, and correctiveoptics configured to focus the optical train on the second emissivecomponent and configured to reduce aberration of detection at the firstor second emissive component. The imaging system further includes aradiation source configured to direct excitation radiation towards thefirst and second emissive components. In addition, the imaging systemincludes detection optics configured to capture emitted radiationreturned from the first and second emissive components via the opticaltrain. Further, the imaging system includes a detector for detecting thecaptured radiation.

DRAWINGS

FIG. 1 is a diagrammatical overview for a biological sample imagingsystem in accordance with the present invention;

FIG. 2 is a diagrammatical perspective view of an exemplary radiationline directed toward a surface of a support structure to semi-confocallyirradiate biological sites, and to semi-confocally return radiation to adetector in accordance with the present invention;

FIG. 3 is a sectional view of an exemplary support structure withexcitation radiation directed at two surfaces of the support structurein accordance with the present invention;

FIG. 4 is a diagrammatical perspective view of an exemplary supportstructure having an array of biological component sites in a spatiallyordered pattern in accordance with the present invention;

FIG. 5 is a diagrammatical perspective view of an exemplary supportstructure having biological component sites in a random spatialdistribution in accordance with the present invention;

FIG. 6 is a sectional view of an exemplary support structure withexcitation radiation directed at multiple surfaces of the supportstructure in accordance with the present invention;

FIG. 7 illustrates exemplary dimensions between the objective and thesupport structure in accordance with the present invention;

FIG. 8 is an exemplary chart of spherical aberration vs. thickness ofthe upper plate of the support structure of FIG. 7 in accordance withthe present invention;

FIG. 9A illustrates exemplary images expected for first and secondsurfaces of a support structure when obtained through an upper surfacethickness of 300 microns (plus 100 microns of fluid) without correctiveoptics, where the imaging system is optimized for the second surface;

FIG. 9B illustrates exemplary images expected for first and secondsurfaces of a support structure when obtained through an upper surfacethickness of 340 microns (plus 100 microns of fluid) without correctiveoptics;

FIG. 10A illustrates an exemplary objective imaging the second surfacewithout the assistance of a compensator in accordance with the presentinvention;

FIG. 10B illustrates an exemplary objective imaging the first surfacewith the assistance of a compensator in accordance with the presentinvention;

FIG. 11 is an exemplary compensator design, incorporating a firstobjective and a second objective which may replace the first objectivein the optical train in accordance with the present invention;

FIG. 12 is another exemplary compensator design, incorporating acorrective device which may be inserted between the objective and thesupport structure in accordance with the present invention;

FIG. 13 is another exemplary compensator design, incorporating acorrection collar in accordance with the present invention;

FIG. 14 is another exemplary compensator design, incorporating aninfinite space compensator in accordance with the present invention;

FIG. 15 is a perspective view of an exemplary flow cell assembly usingpatterned adhesives to form channel characteristics in accordance withthe present invention;

FIG. 16 is a perspective view of another exemplary flow cell assemblyusing patterned adhesives to form channel characteristics in accordancewith the present invention;

FIG. 17 is a process flow diagram of an exemplary method of assemblingflow cells using patterned adhesives to form channel characteristics inaccordance with the present invention;

FIG. 18 is a diagrammatical view of a biological sample imaging systemwith one radiation source and dual detectors configured to sequentiallyscan multiple surfaces of the support structure in accordance with thepresent invention;

FIG. 19 is a diagrammatical view of a biological sample imaging systemwith dual radiation sources and dual detectors configured tosequentially scan multiple surfaces of the support structure inaccordance with the present invention;

FIG. 20 is a diagrammatical view of a biological sample imaging systemwith dual radiation sources and dual detectors configured tosimultaneously scan multiple surfaces of the support structure usingfocusing lenses along the excitation path in accordance with the presentinvention;

FIG. 21 is a diagrammatical view of a biological sample imaging systemwith dual radiation sources and dual detectors configured tosimultaneously scan multiple surfaces of the support structure usingfocusing lenses along the excitation and emission paths in accordancewith the present invention;

FIG. 22 is a diagrammatical view of a biological sample imaging systemwith multiple radiation sources and multiple detectors configured tosimultaneously scan multiple surfaces of the support structure usingfocusing lenses along the excitation and emission paths in accordancewith the present invention;

FIG. 23 is a diagrammatical overview for a TIR biological sample imagingsystem in accordance with the present invention;

FIG. 24 is a sectional view of an exemplary support structure, prism,and lens objective for use with TIR imaging of a bottom surface of aflow lane in accordance with the present invention;

FIG. 25 is a sectional view of an exemplary support structure, prism,and lens objective for use with TIR imaging of a top surface of a flowlane in accordance with the present invention;

FIG. 26 is a sectional view of another exemplary support structure,prism, and lens objective for use with TIR imaging of a top surface of aflow lane in accordance with the present invention; and

FIG. 27 is a sectional view of an exemplary support structure beingheated on both top and bottom surfaces in accordance with the presentinvention.

DETAILED DESCRIPTION

Turning now to the drawings, and referring first to FIG. 1, a biologicalsample imaging system 10 is illustrated diagrammatically. The biologicalsample imaging system 10 is capable of imaging multiple biologicalcomponents 12, 14 within a support structure 16. For instance, in theillustrated embodiment, a first biological component 12 may be presenton a first surface 18 of the support structure 16 while a secondbiological component 14 may be present on a second surface 20 of thesupport structure. The support structure 16 may, for instance, be a flowcell with an array of biological components 12, 14 on the interiorsurfaces 18, 20 which generally mutually face each other and throughwhich reagents, flushes, and other fluids may be introduced, such as forbinding nucleotides or other molecules to the sites of biologicalcomponents 12, 14. The support structure 16 may be manufactured inconjunction with the present techniques or the support structure 16 maybe purchased or otherwise obtained from a separate entity. Fluorescenttags on the molecules that bind to the components may, for instance,include dyes that fluoresce when excited by appropriate excitationradiation. Assay methods that include the use of fluorescent tags andthat can be used in an apparatus or method set forth herein includethose set forth elsewhere herein such as genotyping assays, geneexpression analysis, methylation analysis, or nucleic acid sequencinganalysis.

Those skilled in the art will recognize that a flow cell or othersupport structure may be used with any of a variety of arrays known inthe art to achieve similar results. Furthermore, known methods formaking arrays can be used, and if appropriate, modified in accordancewith the teaching set forth herein in order to create a flow cell orother support structure having multiple surfaces useful in the detectionmethods set forth herein. Such arrays may be formed by disposing thebiological components of samples randomly or in predefined patterns onthe surfaces of the support by any known technique. In a particularembodiment, clustered arrays of nucleic acid colonies can be prepared asdescribed in U.S. Pat. No. 7,115,400; U.S. Patent ApplicationPublication No. 2005/0100900; PCT Publication No. WO 00/18957; or PCTPublication No. WO 98/44151, each of which is hereby incorporated byreference. Such methods are known as bridge amplification or solid-phaseamplification and are particularly useful for sequencing applications.

Other exemplary random arrays, and methods for their construction, thatcan be used in the invention include, without limitation, those in whichbeads are associated with a solid support, examples of which aredescribed in U.S. Pat. Nos. 6,355,431; 6,327,410; and U.S. Pat. No.6,770,441; U.S. Patent Application Publication Nos. 2004/0185483 and US2002/0102578; and PCT Publication No. WO 00/63437, each of which ishereby incorporated by reference. Beads can be located at discretelocations, such as wells, on a solid-phase support, whereby eachlocation accommodates a single bead.

Any of a variety of other arrays known in the art or methods forfabricating such arrays can be used in the present invention.Commercially available microarrays that can be used include, forexample, an Affymetrix® GeneChip® microarray or other microarraysynthesized in accordance with techniques sometimes referred to asVLSIPS™ (Very Large Scale Immobilized Polymer Synthesis) technologies asdescribed, for example, in U.S. Pat. Nos. 5,324,633; 5,744,305;5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716; 5,831,070;5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974,164; 5,981,185;5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269; 6,022,963;6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752; and 6,482,591,each of which is hereby incorporated by reference. A spotted microarraycan also be used in a method of the invention. An exemplary spottedmicroarray is a CodeLink™ Array available from Amersham Biosciences.Another microarray that is useful in the invention is one that ismanufactured using inkjet printing methods such as SurePrint™ Technologyavailable from Agilent Technologies.

Sites or features of an array are typically discrete, being separatedwith spaces between each other. The size of the sites and/or spacingbetween the sites can vary such that arrays can be high density, mediumdensity, or lower density. High density arrays are characterized ashaving sites separated by less than about 15 μm. Medium density arrayshave sites separated by about 15 to 30 μm, while low density arrays havesites separated by greater than 30 μm. An array useful in the inventioncan have sites that are separated by less than 100 μm, 50 μm, 10 μm, 5μm, 1 μm or 0.5 μm. An apparatus or method of the invention can be usedto image an array at a resolution sufficient to distinguish sites at theabove densities or density ranges.

As exemplified herein, a surface used in an apparatus or method of theinvention is typically a manufactured surface. It is also possible touse a natural surface or a surface of a natural support structure;however the invention can be carried out in embodiments where thesurface is not a natural material or a surface of a natural supportstructure. Accordingly, components of biological samples can be removedfrom their native environment and attached to a manufactured surface.

Any of a variety of biological components can be present on a surfacefor use in the invention. Exemplary components include, withoutlimitation, nucleic acids such as DNA or RNA, proteins such as enzymesor receptors, polypeptides, nucleotides, amino acids, saccharides,cofactors, metabolites or derivatives of these natural components.Although the apparatus and methods of the invention are exemplifiedherein with respect to components of biological samples, it will beunderstood that other samples or components can be used as well. Forexample, synthetic samples can be used such as combinatorial libraries,or libraries of compounds having species known or suspected of having adesired structure or function. Thus, the apparatus or methods can beused to synthesize a collection of compounds and/or screen a collectionof compounds for a desired structure or function.

Returning to the exemplary system of FIG. 1, the biological sampleimaging system 10 may include at least a first radiation source 22 butmay also include a second radiation source 24 (or additional sources).The radiation sources 22, 24 may be lasers operating at differentwavelengths. The selection of the wavelengths for the lasers willtypically depend upon the fluorescence properties of the dyes used toimage the component sites. Multiple different wavelengths of the lasersused may permit differentiation of the dyes at the various sites withinthe support structure 16, and imaging may proceed by successiveacquisition of a series of images to enable identification of themolecules at the component sites in accordance with image processing andreading logic generally known in the art. Other radiation sources knownin the art can be used including, for example, an arc lamp or quartzhalogen lamp. Particularly useful radiation sources are those thatproduce electromagnetic radiation in the ultraviolet (UV) range (about200 to 390 nm), visible (VIS) range (about 390 to 770 nm), infrared (IR)range (about 0.77 to 25 microns), or other range of the electromagneticspectrum.

For ease of description, embodiments utilizing fluorescence-baseddetection are used as examples. However, it will be understood thatother detection methods can be used in connection with the apparatus andmethods set forth herein. For example, a variety of different emissiontypes can be detected such as fluorescence, luminescence, orchemiluminescence. Accordingly, components to be detected can be labeledwith compounds or moieties that are fluorescent, luminescent, orchemiluminescent. Signals other than optical signals can also bedetected from multiple surfaces using apparatus and methods that areanalogous to those exemplified herein with the exception of beingmodified to accommodate the particular physical properties of the signalto be detected.

Output from the radiation sources 22, 24 may be directed throughconditioning optics 26, 28 for filtering and shaping of the beams. Forexample, in a presently contemplated embodiment, the conditioning optics26, 28 may generate a generally linear beam of radiation, and combinebeams from multiple lasers, for example, as described in U.S. Pat. No.7,329,860. The laser modules can additionally include a measuringcomponent that records the power of each laser. The measurement of powermay be used as a feedback mechanism to control the length of time animage is recorded in order to obtain uniform exposure, and thereforemore readily comparable signals.

After passing through the conditioning optics 26, 28, the beams may bedirected toward directing optics 30 which redirect the beams from theradiation sources 22, 24 toward focusing optics 32. The directing optics30 may include a dichroic mirror configured to redirect the beams towardthe focusing optics 32 while also allowing certain wavelengths of aretrobeam to pass therethrough. The focusing optics 32 may confocallydirect radiation to one or more surfaces 18, 20 of the support structure16 upon which individual biological components 12, 14 are located. Forinstance, the focusing optics 32 may include a microscope objective thatconfocally directs and concentrates the radiation sources 22, 24 along aline to a surface 18, 20 of the support structure 16.

Biological component sites on the support structure 16 may fluoresce atparticular wavelengths in response to an excitation beam and therebyreturn radiation for imaging. For instance, the fluorescent componentsmay be generated by fluorescently tagged nucleic acids that hybridize tocomplementary molecules of the components or to fluorescently taggednucleotides that are incorporated into an oligonucleotide using apolymerase. As noted above, the fluorescent properties of thesecomponents may be changed through the introduction of reagents into thesupport structure 16 (e.g., by cleaving the dye from the molecule,blocking attachment of additional molecules, adding a quenching reagent,adding an acceptor of energy transfer, and so forth). As will beappreciated by those skilled in the art, the wavelength at which thedyes of the sample are excited and the wavelength at which theyfluoresce will depend upon the absorption and emission spectra of thespecific dyes. Such returned radiation may propagate back through thedirecting optics 30. This retrobeam may generally be directed towarddetection optics 34 which may filter the beam such as to separatedifferent wavelengths within the retrobeam, and direct the retrobeamtoward at least one detector 36.

The detector 36 may be based upon any suitable technology, and may be,for example, a charged coupled device (CCD) sensor that generatespixilated image data based upon photons impacting locations in thedevice. However, it will be understood that any of a variety of otherdetectors may also be used including, but not limited to, a detectorarray configured for time delay integration (TDI) operation, acomplementary metal oxide semiconductor (CMOS) detector, an avalanchephotodiode (APD) detector, a Geiger-mode photon counter, or any othersuitable detector. TDI mode detection can be coupled with line scanningas described in U.S. Pat. No. 7,329,860.

The detector 36 may generate image data, for example, at a resolutionbetween 0.1 and 50 microns, which is then forwarded to acontrol/processing system 38. In general, the control/processing system38 may perform various operations, such as analog-to-digital conversion,scaling, filtering, and association of the data in multiple frames toappropriately and accurately image multiple sites at specific locationson a sample. The control/processing system 38 may store the image dataand may ultimately forward the image data to a post-processing system(not shown) where the data are analyzed. Depending upon the types ofsample, the reagents used, and the processing performed, a number ofdifferent uses may be made of the image data. For example, nucleotidesequence data can be derived from the image data, or the data may beemployed to determine the presence of a particular gene, characterizeone or more molecules at the component sites, and so forth. Theoperation of the various components illustrated in FIG. 1 may also becoordinated with the control/processing system 38. In a practicalapplication, the control/processing system 38 may include hardware,firmware, and software designed to control operation of the radiationsources 22, 24, movement and focusing of the focusing optics 32, atranslation system 40, and the detection optics 34, and acquisition andprocessing of signals from the detector 36. The control/processingsystem 38 may thus store processed data and further process the data forgenerating a reconstructed image of irradiated sites that fluorescewithin the support structure 16. The image data may be analyzed by thesystem itself, or may be stored for analysis by other systems and atdifferent times subsequent to imaging.

The support structure 16 may be supported on a translation system 40which allows for focusing and movement of the support structure 16before and during imaging. The stage may be configured to move thesupport structure 16, thereby changing the relative positions of theradiation sources 22, 24 and detector 36 with respect to the surfacebound biological components for progressive scanning Movement of thetranslation system 40 can be in one or more dimensions including, forexample, one or both of the dimensions that are orthogonal to thedirection of propagation for the excitation radiation line, typicallydenoted as the X and Y dimensions. In particular embodiments, thetranslation system 40 may be configured to move in a directionperpendicular to the scan axis for a detector array. A translationsystem 40 useful in the present invention may be further configured formovement in the dimension along which the excitation radiation linepropagates, typically denoted as the Z dimension. Movement in the Zdimension can also be useful for focusing.

FIG. 2 is a diagrammatical representation of an exemplary semi-confocalline scanning approach to imaging the support structure 16. In theillustrated embodiment, the support structure 16 includes an upper plate42 and a lower plate 44 with an internal volume 46 between the upper andlower plates 42, 44. The upper and lower plates 42, 44 may be made ofany of a variety of materials but may preferably be made of a substratematerial that is substantially transparent at the wavelengths of theexcitation radiation and the fluoresced retrobeam, allowing for thepassage of excitation radiation and returned fluorescent emissionswithout significant loss of signal quality. Moreover, when used inepifluorescent imaging arrangements as shown, one of the surfacesthrough which the radiation traverses may be substantially transparentat the relevant wavelengths, while the other (which is not traversed byradiation) may be less transparent, translucent, or even opaque orreflective. The upper and lower plates 42, 44 may both containbiological components 12, 14 on their respective, inwardly facingsurfaces 18, 20. As discussed above, the internal volume 46 may, forinstance, include one or more internal passages of a flow cell thoughwhich reagent fluids may flow.

The support structure 16 may be irradiated by excitation radiation 48along a radiation line 50. The radiation line 50 may be formed by theexcitation radiation 48 from the radiation sources 22, 24, directed bythe directing optics 30 through the focusing optics 32. The radiationsources 22, 24 may generate beams that are processed and shaped toprovide a linear cross section or radiation line including a pluralityof wavelengths of radiation used to cause fluorescence atcorrespondingly different wavelengths from the biological components 12,14, depending upon the particular dyes used. The focusing optics 32 maythen semi-confocally direct the excitation radiation 48 toward the firstsurface 18 of the support structure 16 to irradiate sites of biologicalcomponent 12 along the radiation line 50. In addition, the supportstructure 16, the directing optics 30, the focusing optics 32, or somecombination thereof, may be slowly translated such that the resultingradiation line 50 progressively irradiates the component as indicated bythe arrow 52. This translation results in successive scanning of regions54 which allow for the gradual irradiation of the entire first surface18 of the support structure 16. As will be discussed in more detailbelow, the same process may also be used to gradually irradiate thesecond surface 20 of the support structure 16. Indeed, the process maybe used for multiple surfaces within the support structure 16.

Exemplary methods and apparatus for line scanning are described in U.S.Pat. No. 7,329,860, which is incorporated herein by reference, and whichdescribes a line scanning apparatus having a detector array configuredto achieve confocality in the scanning axis by restricting the scan-axisdimension of the detector array. More specifically, the scanning devicecan be configured such that the detector array has rectangulardimensions such that the shorter dimension of the detector is in thescan-axis dimension and imaging optics are placed to direct arectangular image of a sample region to the detector array such that theshorter dimension of the image is also in the scan-axis dimension. Inthis way, semi-confocality can be achieved since confocality occurs in asingle axis (i.e. the scan axis). Thus, detection is specific forfeatures on the surface of a substrate, thereby rejecting signals thatmay arise from the solution around the feature. The apparatus andmethods described in U.S. Pat. No. 7,329,860 can be modified such thattwo or more surfaces of a support are scanned in accordance with thedescription herein.

Detection apparatus and methods other than line scanning can also beused. For example, point scanning can be used as described below or inU.S. Pat. No. 5,646,411, which is incorporated herein by reference. Wideangle area detection can be used with or without scanning motion. As setforth in further detail elsewhere herein, TIR methods can also be used.

As illustrated generally in FIG. 2, the radiation line 50 used to imagethe sites of biological components 12, 14, in accordance with thepresent invention, may be a continuous or discontinuous line. As such,some embodiments of the present invention may include a discontinuousline made up of a plurality of confocally or semi-confocally directedbeams of radiation which nevertheless irradiate a plurality of pointsalong the radiation line 50. These discontinuous beams may be created byone or more sources that are positioned or scanned to provide theexcitation radiation 48. These beams, as before, may be confocally orsemi-confocally directed toward the first or second surfaces 18, 20 ofthe support structure 16 to irradiate sites of biological component 12,14. As with the continuous semi-confocal line scanning described above,the support structure 16, the directing optics 30, the focusing optics32, or some combination thereof, may be advanced slowly as indicated byarrow 52 to irradiate successive scanned regions 54 along the first orsecond surfaces 18, 20 of the support structure 16, and therebysuccessive regions of the sites of biological components 12, 14.

It should be noted that the system will typically form and directexcitation and returned radiation simultaneously for imaging. In someembodiments, confocal point scanning may be used such that the opticalsystem directs an excitation point or spot across a biological componentby scanning the excitation beam through an objective lens. The detectionsystem images the emission from the excited point on the detectorwithout “descanning” the retrobeam. This occurs since the retrobeam iscollected by the objective lens and is split off the excitation beamoptical path before returning back through the scan means. Therefore,the retrobeam will appear on the detector 36 at different pointsdepending on the field angle of the original excitation spot in theobjective lens. The image of the excitation point, at the detector 36,will appear in the shape of a line as the excitation point is scannedacross the sample. This architecture is useful, for example, if the scanmeans cannot for some reason accept the retrobeam from the sample.Examples are holographic and acoustic optic scan means that are able toscan a beam at very high speeds but utilize diffraction to create thescan. Therefore, the scan properties are a function of wavelength. Theretrobeam of emitted radiation is at a different wavelength from theexcitation beam. Alternatively or additionally, emission signals may becollected sequentially following sequential excitation at differentwavelengths.

In particular embodiments, an apparatus or method of the invention candetect features on a surface at a rate of at least about 0.01 mm²/sec.Depending upon the particular application of the invention, faster ratescan also be used including, for example, in terms of the area scanned orotherwise detected, a rate of at least about 0.02 mm²/sec, 0.05 mm²/sec,0.1 mm²/sec, 1 mm²/sec, 1.5 mm²/sec, 5 mm²/sec, 10 mm²/sec, 50 mm²/sec,100 mm²/sec, or faster. If desired, for example, to reduce noise, thedetection rate can have an upper limit of about 0.05 mm²/sec, 0.1mm²/sec, 1 mm²/sec, 1.5 mm²/sec, 5 mm²/sec, 10 mm²/sec, 50 mm²/sec, or100 mm²/sec.

In some instances, the support structure 16 may be used in such a waythat biological components are expected to be present on only onesurface. However, in many instances, biological material is present onmultiple surfaces within the support structure 16. For instance, FIG. 3illustrates a typical support structure 16 where biological material hasattached to the first surface 18 as well as to the second surface 20. Inthe illustrated embodiment, an attachment layer 56 has formed on boththe first surface 18 and the second surface 20 of the support structure16. A first excitation radiation 58 source may be used to irradiate oneof many sites of biological component 12 on the first surface 18 of thesupport structure 16 and return a first fluorescent emission 60 from theirradiated biological component 12. Simultaneously or sequentially, asecond source of excitation radiation 62 may be used to irradiate one ofmany sites of biological component 14 on the second surface 20 of thesupport structure 16, and return a second fluorescent emission 64 fromthe irradiated biological component 14.

Although the embodiment exemplified in FIG. 3 shows excitation fromsource 58 and source 62 coming from the same side of the supportstructure 16, it will be understood that the optical system can beconfigured to impinge on the surfaces from opposite sides of the supportstructure 16. Taking FIG. 3 as an example, upper surface 18 can beirradiated from excitation source 58 as shown and the lower surface 20can be irradiated from below. Similarly, emission can be detected fromone or more sides of a support structure. In particular embodiments,different sides of the support structure 16 can be excited from the sameradiation source by first irradiating one side and then flipping thesupport structure to bring another side into position for excitation bythe radiation source.

The distribution of biological components 12, 14 may follow manydifferent patterns. For instance, FIG. 4 illustrates a support structure16 where the biological components 12, 14 at sites or features on thefirst and second surfaces 18, 20 are distributed evenly in a spatiallyordered pattern 66 of biological component sites 68. For example,certain types of microarrays may be used where the location ofindividual biological component sites 68 may be in a regular spatialpattern. The pattern can include sites at pre-defined locations. Incontrast, in other types of biological imaging arrays, biologicalcomponents attach to surfaces at sites that occur in random orstatistically varying positions such that imaging the microarray is usedto determine the location of each of the individual biological componentfeatures. Thus, the pattern of features need not be pre-determineddespite being the product of a synthetic or manufacturing process.

For instance, FIG. 5 illustrates a support structure 16 where the siteson the first and second surfaces 18, 20 are located in a random spatialdistribution 70 of biological component sites 72. However, with bothfixed arrays 66 and random distribution 70 of biological sample sites,imaging of multiple surfaces 18, 20 of the support structure 16 may bepossible. In addition, it should be noted that in both instances, thebiological components at the individual sites may constitute either apopulation of identical molecules or a random mix of differentmolecules. Furthermore, the density of biological samples may vary andmay be at least 1,000 sites per square millimeter.

The present techniques accommodate such varied physical arrangements ofthe multiple surfaces within the support structure 16, as well as thevaried disposition of the sites within components on the surfaces. Asdiscussed above with reference to FIGS. 2 and 3, in the embodiments witha support structure 16 having a first surface 18 and a second surface20, a first source of excitation radiation 58 may irradiate sites ofbiological component 12 on the first surface 18, and return a firstfluorescent emission 60, while a second source of excitation radiation62 may irradiate sites of biological component 14 on the second surface20 and return a second fluorescent emission 64 source, as illustrated inFIG. 3. Thus, components of the volume of sample between two surfacesneed not be detected and can be rejected. Selective detection of asurface of a support structure provides preferential detection of thesurface compared to the volume of the support structure adjacent thesurface and compared to one or more other surfaces of the supportstructure.

In more complex configurations, it may be useful to irradiate more thantwo surfaces. For instance, FIG. 6 illustrates a support structure 16having N number of plates including a first plate 42, a second plate 44,. . . , an N-2 plate 74, an N-1 plate 76, and an N plate 78. Theseplates define M number of surfaces including a first surface 18, asecond surface 20, . . . , an M-3 surface 80, an M-2 surface 82, an M-1surface 84, and an M surface 86. In the illustrated embodiment, not onlythe first surface 18 and the second surface 20 of the support structure16 may be irradiated but, rather, all M number of surfaces may beirradiated. For instance, a source of excitation radiation 88 may beused to irradiate biological component sites on the Mth surface 86 ofthe support structure 16 and return a fluorescent emission 90 from theirradiated biological component. For support structures having aplurality of surfaces it may be desirable to excite upper layers fromthe top and lower layers from the bottom to reduce photobleaching. Thus,components on layers that are closer to a first exterior side of asupport structure can be irradiated from the first side, whereasirradiation from the opposite exterior side can be used to excitecomponents present on layers that are closer to the opposite exteriorside.

FIG. 7 illustrates an objective 92 through which radiation from emissivebiological components 12, 14 on first and second surfaces 18, 20,respectively, of the support structure 16 may be detected. The objective92 may be one of the components of the focusing optics 32 describedabove. Although not drawn to scale, FIG. 7 illustrates exemplarydimensions between the objective 92 and the support structure 16. Forinstance, the objective 92 may typically be spaced approximately 600 ormore microns from the upper plate 42 of the support structure 16. Thebiological sample imaging system 10 may be configured to detect emittedradiation from biological components 12 on the first surface 18 through300 microns of the upper plate 42 which may, for instance, be made ofglass and may have a refractive index N_(d) of 1.472. In addition, thebiological sample imaging system 10 may also be configured to detectemitted radiation from biological components 14 on the second surface 20through 300 microns of the upper plate 42 plus 100 microns of the fluidwithin the internal volume 46 of the support structure 46.

In certain embodiments, the objective 92 may be designed fordiffraction-limited focusing and imaging on only one of the first orsecond surfaces 18, 20 of the support structure 16. For examplethroughout the present description of FIGS. 7 through 14, the objective92 may be designed for pre-compensation of the 300 microns of the upperplate 42 plus the 100 micron read buffer of the fluid within theinternal volume 46 of the support structure 16. In such a scenario,diffraction-limited performance may only be possible on the secondsurface 20. Furthermore, the spherical aberration introduced by the 100micron read buffer may severely impact the imaging quality when imagingfrom the first surface 18. However, reducing the lane thickness of theinternal volume 46 of the support structure 16 might increase the amountof surface-to-surface “crosstalk.” Therefore, perhaps the mostappropriate solution is to correct the aberration. As such, it may benecessary to use a compensator capable of achieving diffraction-limitedimaging performance on both the first and second surfaces 18, 20 of thesupport structure 16.

It should be noted that the need for a compensator may be morepronounced when using objectives 92 with high numerical aperture (NA)values. Exemplary high NA ranges for which the invention is particularlyuseful include NA values of at least about 0.6. For example, the NA maybe at least about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher.Those skilled in the art will appreciate that NA, being dependent uponthe index of refraction of the medium in which the lens is working, maybe higher including, for example, up to 1.0 for air, 1.33 for purewater, or higher for other media such as oils. The compensator may alsofind use in objectives having lower NA values than the examples listedabove. In general, the NA value of an objective 92 is a measure of thebreadth of angles for which the objective 92 may receive light. Thehigher the NA value, the more light that may be collected by theobjective 92 for a given fixed magnification. This is because thecollection efficiency and the resolution increase. As a result, multipleobjects may be distinguished more readily when using objectives 92 withhigher NA values because a higher feature density may be possible.Therefore, in general, a higher NA value for the objective 92 may bebeneficial for imaging. However, as the NA value increases, itssensitivity to focusing and imaging-through media thickness variationalso increases. In other words, lower NA objectives 92 have longer depthof field and are generally not as sensitive to changes inimaging-through media thickness.

FIG. 8 is an exemplary chart 94 of spherical aberration (in waves) vs.thickness (in microns) of the upper plate 42 of the support structure 16of FIG. 7 in accordance with the present invention. Specifically, theupper line 96 of the graph depicts the amount of spherical aberration ofan image taken from biological components 12 on the first surface 18 ofthe support structure 16 while the lower line 98 of the graph depictsthe amount of spherical aberration of an image taken from biologicalcomponents 14 on the second surface 20 of the support structure 16. Inthe illustrated embodiment, the spherical aberration generated by the100 micron read buffer is around 4 waves, which is much higher than thediffraction-limited performance requirement of less than 0.25 waves, forinstance. As illustrated, at 300 microns (i.e. the thickness of theupper plate 42), the spherical aberration for the first surface 18 isaround −13.2 waves (e.g., point 100) while the spherical aberration forthe second surface 20 is around −17.2 waves (e.g., point 102). FIG. 9Aillustrates exemplary images expected for the first and second surfaces18, 20 of the support structure 16 corresponding to the thickness of theupper plate 42 (i.e., 300 microns) in accordance with the presentinvention, where the imaging system is optimized for the second surface20 (pre-compensated for −17.2 waves). As shown, the imaging system iscapable of providing high image quality on the second surface 20 since,according to the present scenario, it was designed to do so. However,the images taken for the first surface 18 contain aberrations.

To balance out the spherical aberration, it is beneficial to introducean additional thickness (e.g., by introducing an additional coverslip)between the objective 92 and the support structure 16. For instance,returning now to FIG. 8, if an additional thickness of approximately 40microns were to be introduced between the objective 92 and the first andsecond surfaces 18, 20 of support structure 16, the difference betweenthe spherical aberrations at the design thickness (i.e., 300 micronupper plate plus 100 microns of fluid) may be split such that the imageproduced for both the first and second surfaces 18, 20 may have similarquality. For instance, as illustrated, at 340 microns (i.e. thethickness of the upper plate 42 plus an additional 40 micron thickness),the spherical aberration for the first surface 18 is around −15.2 waves(e.g., point 104) while the spherical aberration for the second surface20 is around −19.2 waves (e.g., point 106), splitting the difference of−17.2 waves (e.g., point 108) which may be characterized as the designpoint for the objective 92. FIG. 9B illustrates exemplary imagesexpected for the first and second surfaces 18, 20 of the supportstructure 16 corresponding to the thickness of the upper plate 42 plusan additional thickness (i.e., 300 microns plus 40 microns) inaccordance with the present invention, illustrating how the additionalthickness may allow for balance between images taken for the first andsecond surfaces 18, 20 of the support structure 16.

However, merely introducing an additional thickness between theobjective 92 and the support structure 16 may not be desired for alluses of the imaging system set forth herein. For instance, asillustrated in FIGS. 9A and 9B, by simply introducing the additional 40micron thickness between the objective 92 and the support structure 16,images from both the first and second surfaces 18, 20 may stillexperience residual aberration from the design point 108 of theobjective 92. Therefore, a more precise solution may be to onlyintroduce the additional thickness when detecting radiation frombiological components 12 on the first surface 18 of the supportstructure 16. In such a scenario, the spherical aberration correspondingto the design point 108 of the objective 92 may generally be achievedfor both the first and second surfaces 18, 20. It should be noted thatthe particular dimensions and measurements (e.g., thicknesses, sphericalaberration values, and so forth) described with respect to FIGS. 9A and9B are merely intended to be exemplary of the manner in which thepresent invention functions. As such, these dimensions and measurementsare not intended to be limiting. Indeed, the particular geometries andresulting measurement values may vary between implementations.

For example, FIG. 10A illustrates an exemplary objective 92 imaging thesecond surface 20 of the support structure 16 without the assistance ofa compensator 110 in accordance with the present invention. Without thecompensator 110, the objective 92 may focus and detect images from thesecond surface 20 of the support structure 16 according to its designand experiencing the design spherical aberration. However, FIG. 10Billustrates an exemplary objective 92 imaging the first surface 20 ofthe support structure 16 with the assistance of a compensator 110 inaccordance with the present invention. By using the compensator 110(e.g., similar to the additional 40 micron thickness described abovewith respect to FIGS. 8 and 9), the objective 92 may focus and detectimages from the first surface 18 of the support structure 16 undersimilar conditions to that of its design point for the second surface 20of the support structure 16. Therefore, by detecting images for thesecond surface 20 without the compensator 110 and detecting images forthe first surface 18 with the compensator 110, the objective 92 may becapable of detecting images on both surfaces with diffraction-limitedperformance similar to the design of the objective 92.

The chromatic shift curve may be limited to wavelength ranges of between530 nm to 780 nm. Chromatic shifts of different color wavelength bandsmay be compensated for by focusing the focusing optics 32 in each band.The compensator 110 should preferably be “invisible” to the focusingoptics 32. In other words, the compensator 110 should correct thespherical aberration difference of the read buffer but should maintainthe chromatic shift curve in the wavelength range of 530-780 nm. Morespecifically, the chromatic shift relationships among the peakwavelengths of 560 nm, 610 nm, 687 nm, and 720 nm should be maintained.In addition, other specifications, including NA, field curvature, fielddistortion, detection magnification, and so forth, should also bemaintained. Furthermore, the compensator 110 package should berelatively small (e.g., no more than 10 mm of total thickness).Moreover, insensitivity to positioning error of the compensator 110 maybe preferred.

Several various designs may be implemented to introduce the correctiveoptics of the compensator 110 into the optical train of the imagingoptics of the biological sample imaging system 10. For example, FIG. 11is an exemplary compensator 110 design, incorporating a first objective92 and a second objective 112 which may replace the first objective 92in the optical train in accordance with the present invention. In theillustrated embodiment, each respective objective 92, 112 may containthe optics required to image respective surfaces, such as the first andsecond surfaces 18, 20 of the support structure 16. For instance, thefirst objective 92 may contain the imaging optics necessary to focus onand image emissive biological components 14 on the second surface 20 ofthe support structure 16 while the second objective 112 may contain theimaging optics plus the corrective optics necessary to focus on andimage emissive biological components 12 on the first surface 18 of thesupport structure 16. In operation, the first objective 92 may detectimages from the second surface 20 of the support structure 16. The firstobjective 92 may be replaced by the second objective 112 in the opticaltrain, at which point the second objective 112 may detect images fromthe first surface 18 of the support structure 16. An advantage of theembodiment illustrated in FIG. 11 is that the optics may be decoupledand may operate independently. However, a disadvantage in somesituations is that having two entirely separate objectives 92, 112 maynot be cost-effective since certain components may be duplicated foreach objective 92, 112. Furthermore, in embodiments where multipleimages of an object are obtained, the use of two objectives may increasethe computational resources required for registration between images. Inparticular embodiments, imaging of both surfaces may occur through thesame objective to provide particular advantages as set forth below. Inother words, the first objective 92 need not be removed or replaced withthe second objective 112 for imaging of the different surfaces.

FIG. 12 is another exemplary compensator 110 design, incorporating acorrective device 114 which may be inserted between the objective 92 andthe support structure 16 in accordance with the present invention. Thecorrective device 114 may, for instance, be a coverslip or other thinlayer of glass. As illustrated, the corrective device 114 may simply beinserted into and removed from the optical path between the objective 92and the support structure 16 depending on the particular surface 16being imaged. For instance, the corrective device 114 may be removedfrom the optical path when the objective 92 is used to focus on andimage emissive biological components 14 on the second surface 20 of thesupport structure 16. Conversely, the corrective device 114 may beinserted into the optical path when the objective 92 is used to focus onand image emissive biological components 12 on the first surface 18 ofthe support structure 16. An advantage of the embodiment illustrated inFIG. 12 is that it is relatively straightforward. The requiredadditional compensator thickness may simply be inserted into the opticalpath. Typically, the corrective device 114 may be placed such that itdoes not physically contact the support structure 16 or the objective92.

FIG. 13 is another exemplary compensator 110 design, incorporating acorrection collar 116 in accordance with the present invention. In theillustrated embodiment, the correction collar 116 may be adjustedbetween binary states. For instance, the first state 118 may correspondto the situation where the objective 92 is focused on and detectingimages from the second surface 20 of the support structure 16 while thesecond state 120 may correspond to the situation where the objective 92is focused on and detecting images from the first surface of the supportstructure 16. As such, when the correction collar 116 is in the firststate 118, the imaging optics within the objective 92 may not includethe corrective optics within the optical path. Conversely, when thecorrection collar 116 is in the second state 120, the imaging opticswithin the objective 92 may include the corrective optics within theoptical path. Although illustrated as consisting of binary states 118,120, the correction collar 116 may, in fact, include multiple states.For instance, when more than two surfaces of the support structure 16are used for imaging, the correction collar 116 may be configured toadjust between multiple states such that the imaging and correctiveoptics vary for each respective surface of the support structure 16. Anadvantage of the embodiment illustrated in FIG. 13 is that it may berelatively easy to operate. For instance, the correction collar 116 maysimply be adjusted between states whenever different surfaces of thesupport structure 16 are being imaged.

FIG. 14 is another exemplary compensator 110 design, incorporating aninfinite space compensator 122 in accordance with the present invention.This embodiment is somewhat similar to the corrective device 114embodiment of FIG. 12 in that the infinite space compensator 122 may beinserted into and removed from the optical path. However, a maindifference between the embodiments is that, in the embodiment of FIG.14, there may be more space available (e.g., up to 10 mm, as opposed to600 microns in the embodiment of FIG. 12) within which to insert theinfinite space compensator 122 into the optical path. Therefore, theembodiment of FIG. 14 may allow for greater flexibility than thecorrective device 114 embodiment of FIG. 12.

In addition to the embodiments presented in FIGS. 11 through 14, theremay be other compensator 110 designs which may prove beneficial. Forinstance, a fluidic corrector may be inserted between the objective 92and the support structure 16. In this fluidic corrector design, thefluidic corrector may be filled with a fluid, which may effectively actas the compensator 110. The optics may be configured such that the fluidmatches the upper surface of the support structure 16 and, in theabsence of fluid air, matches the bottom surface of support structure16. This design may prove beneficial in that it may make automationeasier since the fluid would simply be inserted into and extracted fromthe fluidic corrector depending on which surface is imaged.

Regardless of the particular embodiment selected, all of the embodimentsdisclosed herein are characterized by repeatability and the ability toautomate the use of the embodiments. These are important considerationsin that the embodiments allow for the detection of images frombiological components 12, 14 on multiple surfaces 18, 20 of the supportstructure 16 in an automated fashion. This may allow not only forincreased imaging production but may also allow for greater flexibilityin switching between the multiple surfaces, depending on the particularimaging needs.

As described in greater detail above, a support structure 16 useful inthe apparatus or methods set forth herein can have two or more surfacesupon which a biological component is attached. In particularembodiments, the surface is a fabricated surface. Any of a variety ofsurfaces known in the art can be used including, but not limited to,those used for making arrays as set forth above. Examples include,glass, silicon, polymeric structures, plastics, and the like. Surfacesand flow cells that are particularly useful are described in PCTPublication No. WO 2007/123744, which is incorporated herein byreference. The surfaces of a support structure can have the same ordifferent properties. For example, in the embodiment shown in FIG. 3,plate 42 can be transparent to the excitation and emission wavelengthsused in a detection method, whereas plate 44 can optionally betransparent or opaque to the excitation or emission wavelengths.Accordingly, the surfaces can be made of the same material or the two ormore surfaces can be made of different materials.

A support structure having two or more surfaces can be formed byadhering the surfaces to each other or to other supports. For example,an adhesive material, such as epoxy resin, can be dispensed in the formof a paste onto a planar substrate in a pattern forming one or morechannel characteristics of a flow cell. An exemplary flow cell 124 isshown in FIG. 15. Utilizing a programmable, automated adhesivedispenser, such as the Millennium® M-2010 from Asymtek Corp., CarlsbadCalif., a desired pattern of adhesive 126 can be designed and laid downonto the surface of a planar lower substrate 128. The thickness of theflow cell (and cross sectional height in the fluidic channels) can beset by means of precision mechanical spacers 130 placed between thelower substrate 128 and an upper substrate 132. Another exemplary flowcell 134 is shown in FIG. 16. To create a multi-layer cell, an interimtransparent substrate layer 136, shorter in length than the lower andupper substrate layers 128, 132 can be included. The shorter lengthallows fluidic access to both/all layers from ports 138 passing throughonly one substrate. This intermediate layer 136 bifurcates the flow cellcavity horizontally and nearly doubles the available surface area forthe attachment of biologically interesting molecules.

An exemplary method 140 for fabricating such a flow cell is shown inFIG. 17. A planar substrate acting as the structural base of the cell isprovided (block 142). Desired canalizing features of the cell aredesigned, for example, using a computer assisted design program (block144). A pattern designed in this way can be exported to a filecompatible with driving an automatic adhesive dispensing system (block146). A program can be executed to dispense the adhesive in the desiredpattern onto the substrate (block 148). Precision mechanical spacers canbe placed onto the base substrate before or after the adhesive isdispensed (block 150). A second transparent substrate can then be placedonto the adhesive pattern, pressing downward until the lower surface isin full contact with the mechanical spacers (block 152). A weight orother force is applied to the top substrate to hold it in full contactwith the adhesive. The spacers will typically have a height that isequivalent or slightly less than the height of the adhesive layer suchthat bonding can occur without causing undesirable aberrations in theshape of the canalized features. The steps for adhering substrates maybe repeated for any number of layers desired. Optionally, the assemblycan be heat treated, for example, in an oven or exposed to UV light,depending upon the cure requirements of the adhesive (block 154).

Another exemplary method for fabricating a flow cell is to use anintermediate layer that is cut to a desired pattern in place of anadhesive layer. A particularly useful material for the intermediatelayer is silicone. The silicone layer can be heat bonded to the lowersubstrate 128 and upper substrate 132. Exemplary methods utilizing BiscoSilicone HT 6135 as an intermediate layer are described, for example, inGrover et al., Sensors and Actuators B 89:315-323 (2003).

Still further, FIG. 18 illustrates an embodiment utilizing one radiationsource and dual detectors. Radiation from the radiation source 22 isdirected by the directing optics 30 toward the focusing optics 32. Fromthe focusing optics 32, the excitation radiation 58 irradiates abiological component 12 on a first surface 18 of the support structure16. The biological component 12 emits a fluorescent emission 60 backthrough the focusing optics 32 toward the directing optics 30. Thisretrobeam is allowed to pass through the directing optics 30 to thedetection optics 34 which, in this illustrated embodiment, may include awavelength filter 156 or some other device for separating the retrobeam,and first and second color filters 158, 160 for achieving multiple colorchannels. The wavelength filter 156 may split the retrobeam into twobeams with one beam directed toward the first detector 36 via the firstcolor filter 158 and the other beam directed toward a second detector162 via the second color filter 160. In this manner, the biologicalsample imaging system 10 may sequentially scan the first and secondsurfaces 18, 20, first scanning the first surface 18 of the supportstructure 16 using the first excitation radiation 58 from the radiationsource 22 and the returned first fluorescent emission 60 (as depicted inthe left portion of FIG. 18), and next scanning the second surface 20 ofthe support structure 16 using the second excitation radiation 62 fromthe same radiation source 22 and the returned second fluorescentemission 64 (as depicted in the right portion of FIG. 18).

Alternatively, FIG. 19 illustrates an embodiment utilizing dualradiation sources and dual detectors. Again, the two surfaces 18, 20 ofthe support structure 16 may be scanned sequentially. However, in thisembodiment, the first surface 18 of the support structure 16 is firstscanned using the first radiation source 22 which generates the firstexcitation radiation 58 and the first fluorescent emission 60 (asdepicted in the left portion of FIG. 19) and, the second surface 20 ofthe support structure 16 is scanned using the second radiation source 24which generates the second excitation radiation 62 and the secondfluorescent emission 64 (as depicted in the right portion of FIG. 19).This embodiment may also be extended to use any number of detectors inorder to reduce movement of the filters.

In the embodiments described above where scanning of the first andsecond surfaces 18, 20 of the support structure 16 may be performedsequentially, the individual steps of scanning the first and secondsurfaces 18, 20 of the support structure 16 may be performed in a numberof ways. For instance, it may be possible to scan a single line of thefirst surface 18, then scan a single line of the second surface 20, thengradually move the first and second surfaces 18, 20 relative to theexcitation radiation 58, 62 by translating the support structure 16, thedirecting optics 30, the focusing optics 32, or some combinationthereof, in order to repeat these steps of scanning individual lines.Alternatively, entire regions of the first surface 18 may be scannedbefore regions of the second surface 20 are scanned. The individualprocessing steps taken may depend upon several variables including theparticular configuration of the biological component sites 12, 14 on thesurfaces 18, 20 as well as other variables, including environmental andoperating conditions.

Particular embodiments may allow for simultaneous excitation of multiplesurfaces of the support structure 16. For instance, FIG. 20 illustratesan embodiment utilizing dual radiation sources and dual detectors.However, in this embodiment, the first surface 18 and the second surface20 of the support structure 16 may be simultaneously scanned. This maybe accomplished using focusing lenses 164, 166, 168, 170 and a dichroicmirror 172 along the excitation path in order to switch surfaces andfilters 158, 160 to achieve multiple color channels. Again, thisillustrated embodiment may also be extended to any number of detectorsto improve throughput, scanning efficiency, and to reduce movement ofthe filters and other system components.

FIG. 21 illustrates another embodiment utilizing dual radiation sourcesand dual detectors which allows for simultaneous scanning of the firstand second surfaces 18, 20 of the support structure 16. In thisillustrated embodiment, however, not only are focusing lenses 164, 166,168, 170 and a dichroic mirror 172 used in the excitation path butfocusing lenses 174, 176 may be used just upstream of the first andsecond detectors 36, 162 in conjunction with the filters 158, 160 alongthe emission path in order to switch surfaces and achieve multiple colorchannels. Once again, this illustrated embodiment may also be extendedto use any number of detectors to increase throughput and scanningefficiency.

For instance, FIG. 22 illustrates an embodiment utilizing multipleradiation sources and multiple detectors which are capable ofsimultaneously outputting multiple channels with few moving parts. Inthe illustrated embodiment, radiation sources 22 and 24 have beenreplaced by radiation source groups 178 and 180 which are capable ofoutputting multiple radiation sources and varying wavelengths. Inaddition, detectors 36 and 38 have been replaced by detector groups 182and 184 in the illustrated embodiment. These detector groups 182, 184are similarly capable of detecting multiple color channels. Thisembodiment therefore illustrates the considerable adaptability of thepresent techniques to a range of configurations capable of imagingcomponents on multiple surfaces of the support.

In the embodiments described above where scanning of the first andsecond surfaces 18, 20 of the support structure 16 may be performedsimultaneously, focusing of the excitation radiation 58 source may beaccomplished in several various ways. For instance, it may be possibleto focus the excitation radiation 58 on one of the surfacespreferentially over the other surface. In fact, due to the nature of theconfiguration of the first surface 18 with respect to the second surface20, it may be necessary to do so. However, alternate focusing techniquesmay be employed depending on the specific configuration of the supportstructure 16. Moreover, it may be advantageous in these variousconfigurations to first image the upper surface (i.e., the surfacecloser to the radiation source) in order to reduce photobleaching of thecomponents on that surface that could result from first imaging thelower surface (i.e., the surface farther from the radiation source).Such selection of which surface to image may apply both when thesurfaces are imaged sequentially as well as when they are imagedsimultaneously.

In addition, the embodiments disclosed above have illustrated anepifluorescent imaging scheme wherein the excitation radiation isdirected toward the surfaces of the support structure 16 from a topside, and returned fluorescent radiation is received from the same side.However, the techniques of the present invention may also be extended toalternate arrangements. For instance, these techniques may also beemployed in conjunction with TIR imaging whereby the surfaces of thesupport structure are irradiated from a lateral side with radiationdirected at an incident angle within a range of critical angles so as toconvey the excitation radiation within the support or into the supportfrom a prism positioned adjacent to it. TIR techniques can be carriedout as described, for example, in U.S. Patent Application PublicationNo. 2005/0057798, which is hereby incorporated by reference. Suchtechniques cause fluorescent emissions from the components that areconveyed outwardly for imaging, while the reflected excitation radiationexits via a side opposite from that through which it entered. Hereagain, biological components on the multiple surfaces may be imagedsequentially or simultaneously.

For example, in FIG. 23, a TIR biological sample imaging system 186 isillustrated diagrammatically. A support structure 188 may be used whichincludes multiple flow lanes 190 containing biological components. Forexample, the support structure 188 may be a flow cell through whichreagents, flushes, and other fluids may be introduced using the flowlanes 190 to contact emissive components attached to the surface of theflow cell. The support structure 188 may be supported by a prism 192. Inthe TIR biological sample imaging system 186, the radiation source 194may output a radiation beam 196 through the prism 192 from a lateralside of the support structure 188. The radiation beam 196 may, forinstance, be directed toward a bottom surface of one of the flow lanes190 of the support structure 188, thereby exciting emissive componentsattached to the surface.

As discussed in further detail below, as long as the incident angle ofthe radiation beam 196 is within the range of critical angles (asdescribed, for example, in US 2005/0057798), a portion of the radiationbeam 196 will be reflected off the bottom surface whereas a separatefluorescent emission beam from surface-bound emissive components will bedirected toward focusing optics 198. Typically, a well collimatedradiation beam is used to prevent spread of angles within the beam,thereby preventing unwanted hindrance of total internal reflectance. Thefluorescent emission beam may propagate back through the focusing optics198, directing optics 200, and detection optics 202 which may direct thebeam toward a detector 204. The focusing optics 198, directing optics200, detection optics 202, and detector 204 may operate in much the samemanner as with the epifluorescent techniques discussed above. In the TIRbiological sample imaging system 186, the focusing light source 206 maybe used as a separate light source from the radiation source 194 tofocus the optics on a particular surface to be imaged. For instance, thefocusing light source 206 may be directed to the directing optics 200where it is redirected toward the focusing optics 198 which are used tofocus the system on a particular surface of the support structure 188.

The TIR biological sample imaging system 186 may also include atranslation system 208 for moving the support structure 188 and prism192 in one or more dimensions. The translation system 208 may be usedwith focusing, redirecting the radiation source 194 to different areasof the support structure 188, as well as for moving the supportstructure 188 and prism 192 to a heating/cooling station 210. Theheating/cooling station 210 may be used to heat and cool the supportstructure 188 before and after imaging. In addition, acontrol/processing system 212 may be used to control operation of theradiation source 194, the focusing light source 206, and theheating/cooling station 210, movement and focusing of the focusingoptics 198, the translation system 208, and the detection optics 202,and acquisition and processing of signals from the detector 204.

As discussed above, the TIR method of imaging may be used to direct theradiation beam 196 from a lateral side of the support structure 188, asillustrated in FIG. 24. Each flow lane 190 of the support structure 188may include a bottom surface 214 and a top surface 216 and emissivecomponents can optionally be attached to either or both surface. In theillustrated embodiment, the radiation beam 196 is directed toward abottom surface 214 of one of the flow lanes 190 of the support structure188. Part of the radiation beam 196 may be reflected off the bottomsurface 214 of the flow lane 190, as depicted by reflected light beam218. However, as long as the incident angle of the radiation beam 196 iswithin the range of critical angles, a separate fluorescent emissionbeam 220 may be emitted from emissive components toward the focusingoptics 198 which in the illustrated embodiment is a lens objective 222.Indeed, directing the radiation beam 196 at a bottom surface 214 of aflow lane 190 of the support structure 188 is a typical implementationof the TIR imaging method. However, in doing so, imaging data which maybe collected from a top surface 216 of a flow lane 190 of the supportstructure 188 may be overlooked.

Therefore, the orientation of the radiation source 194 and/or thesupport structure 188 and prism 192 may be adjusted in order to allowthe radiation beam 196 to not be directed at a bottom surface 214 of aflow lane 190 of the support structure 188, as illustrated in FIG. 25.In the illustrated embodiment, the radiation beam 196 is oriented sothat the radiation beam 196 passes through the prism 192 and supportstructure 188 until contacting an air/glass interface 224 of the supportstructure 188 at which point the radiation beam 196 is redirected towarda top surface 216 of a flow lane 190 of the support structure 188. Atthis point, part of the radiation beam 196 may be reflected back towardanother air/glass interface 224 of the support structure 188. However, aseparate fluorescent emission beam 220 may be emitted from an emissivecomponent on the top surface 216 toward the lens objective 222. Usingthis technique, top surfaces 216 of the flow lanes 190 of the supportstructure 188 may be imaged using TIR imaging methods. This, in effect,may allow for double the imaging data output for cluster basedsequencing applications while keeping other variables, such as surfacecoating, cluster creation, and sequencing, the same.

In order to accomplish this TIR imaging of top surfaces 216 of the flowlanes 190 of the support structure 188, the radiation beam 196 reachesthe air/glass interface 224 of the support structure 188 unperturbed. Todo so, the radiation beam 196 does not first come into contact withemissive components in adjacent flow lanes 190. To do so, either theradiation beam 196 may be directed around the adjacent flow lanes 190 orthe adjacent flow lanes 190 may be index matched with the supportstructure 188 material. In some embodiments, the flow lanes 190 may bespaced within the support structure 188, leaving sufficient room betweenthe flow lanes 190 for the radiation beam 196 to pass. However, spacingthe flow lanes 190 in this manner may ultimately reduce the amount ofemissive components which may be imaged. Therefore, in otherembodiments, it may be possible to accomplish the same effect bytemporarily filling alternate flow lanes 190 with index matching fluid.Doing so may allow for easier direction of the radiation beam 196 towarda top surface 216 of a flow lane 190 of the support structure 188.

It may also be possible to direct the radiation beam 196 in such a waythat it bounces off multiple top surfaces 216 of flow lanes 190 of thesupport structure 188, as illustrated in FIG. 26. In order to accomplishthis, the spacing of the flow lanes 190 can be matched with the angle ofradiation beam 196 such that the radiation beam 196 is able to pass bythe flow lanes 190, such that it reaches the air/glass interface 224 ofthe support structure 188 unperturbed, while also being able to bounceback and forth between top surfaces 216 of flow lanes 190 and theair/glass interface 224 of the support structure 188. As describedabove, in certain embodiments, some of the flow lanes 190 may be filledwith an index matching fluid, such that these index-matched flow lanes190 effectively become “invisible” to the radiation beam 196. In otherwords, the radiation beam 196 may be allowed to pass through theindex-matched flow lanes 190. By allowing the radiation beam 196 to passthrough the index-matched flow lanes 190, the support structure 188 maybe used in multiple configurations without the need of varying thespacing of the flow lanes 190.

In some embodiments, mirrors 226 or other suitable reflective materialmay be used within certain flow lanes 190, facilitating thismulti-bounce technique. In any event, assuming N number of flow lanes190, it may only be possible to image N-2 number of top surfaces 216 ofthe flow lanes 190 in this manner due to the fact that the outer flowlanes 190 on either side of the support structure 188 may not beaccessible using these techniques. However, modification of the prism192 and/or support structure 188 may allow for imaging of the topsurfaces 216 of these outermost flow lanes 190. For instance, thesupport structure 188 may be designed to fit within the prism 192,allowing the radiation beam 196 to propagate into a lateral side of thesupport structure 188.

In some embodiments, as discussed above briefly with respect to FIG. 23,the support structure 188 may be moved to a heating/cooling station 210,for example, by the action of the translation system 208. Theheating/cooling station 210 may be configured to both heat and cool thesupport structure 188 before and after imaging. The heating/coolingstation 210 may, in fact, be configured to heat and cool both a topsurface 228 and a bottom surface 230 of the support structure 188, asillustrated in FIG. 27. Indeed, all surfaces of the support structure188 may be heated or cooled at the heating/cooling station 210. In thismanner, it may further be possible to heat and cool both the topsurfaces 216 and bottom surfaces 214 of the flow lanes 190 of thesupport structure 188 by directly contacting one or more surfaces of theflow cell with a heating or cooling device. This, of course, mayfacilitate the development of biological components within the flowlanes 190 of the support structure 188 and, therefore, facilitateimaging. Although use of the heating/cooling station 210 has beenpresented herein with respect to the TIR imaging methods, theheating/cooling station 210 may also be used to heat and cool multiplesides of a support structure used in conjunction with the epifluorescentimaging methods discussed herein.

In particular embodiments, the current invention utilizessequencing-by-synthesis (SBS). In SBS, four fluorescently labeledmodified nucleotides are used to determine the sequence of nucleotidesfor nucleic acids present on the surface of a support structure such asa flow cell. Exemplary SBS systems and methods which can be utilizedwith the apparatus and methods set forth herein are described in U.S.Pat. No. 7,057,026; U.S. Patent Application Publication Nos.2005/0100900, 2006/0188901, 2006/0240439, 2006/0281109, and2007/0166705; and PCT Publication Nos. WO 05/065814, WO 06/064199, andWO 07/010251; each of which is incorporated herein by reference.

In particular uses of the apparatus and methods herein, flow cellscontaining arrayed nucleic acids are treated by several repeated cyclesof an overall sequencing process. The nucleic acids are prepared suchthat they include an oligonucleotide primer adjacent to an unknowntarget sequence. To initiate the first SBS sequencing cycle, one or moredifferently labeled nucleotides and a DNA polymerase are flowed into theflow cell. Either a single nucleotide can be added at a time, or thenucleotides used in the sequencing procedure can be specially designedto possess a reversible termination property, thus allowing each cycleof the sequencing reaction to occur simultaneously in the presence ofall four labeled nucleotides (A, C, T, G). Following nucleotideaddition, the features on the surface can be imaged to determine theidentity of the incorporated nucleotide (based on the labels on thenucleotides). Then, reagents can be added to the flow cell to remove theblocked 3′ terminus (if appropriate) and to remove labels from eachincorporated base. Such cycles are then repeated and the sequence ofeach cluster is read over the multiple chemistry cycles.

Other sequencing methods that use cyclic reactions wherein each cycleincludes steps of delivering one or more reagents to nucleic acids on asurface and imaging the surface bound nucleic acids can also be usedsuch as pyrosequencing and sequencing by ligation. Useful pyrosequencingreactions are described, for example, in U.S. Pat. No. 7,244,559 andU.S. Patent Application Publication No. 2005/0191698, each of which isincorporated herein by reference. Sequencing by ligation reactions aredescribed, for example, in Shendure et al. Science 309:1728-1732 (2005);and U.S. Pat. Nos. 5,599,675 and 5,750,341, each of which isincorporated herein by reference.

The methods and apparatus described herein are also useful for detectionof features occurring on surfaces used in genotyping assays, expressionanalyses and other assays known in the art such as those described inU.S. Patent Application Publication Nos. 2003/0108900, 2003/0215821, andUS 2005/0181394, each of which is incorporated herein by reference.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

The invention claimed is:
 1. A system, comprising: a flow cellcomprising first and second surfaces separated by a fluid passagethrough which a fluorescent reagent flows to add fluorescent tags tonucleic acid sites distributed on the first and second surfaces; and adetection system configured to detect the fluorescent tags todistinguish the nucleic acid sites on the first surface, and to detectthe fluorescent tags to distinguish the nucleic acid sites on the secondsurface.
 2. The system of claim 1, wherein the nucleic acid sites aredistributed in a spatially ordered pattern on the first surface and onthe second surface.
 3. The system of claim 1, wherein the nucleic acidsites are distributed in a random spatial distribution on the firstsurface and on the second surface.
 4. The system of claim 1, wherein thenucleic acid sites are separated with spaces between each other.
 5. Thesystem of claim 1, wherein the nucleic acid sites are present at adensity of at least 1,000 sites per square millimeter.
 6. The system ofclaim 1, wherein the detection system is configured to detect thefluorescent tags at an optical resolution between 0.1 and 50 microns. 7.The system of claim 1, wherein the flow cell is configured to betranslated, and the detection system if configured to detect thefluorescent tags at successive regions of the first surface and atsuccessive regions of the second surface, respectively.
 8. The system ofclaim 7, wherein the detection system is configured to perform wideangle area detection of each successive region.
 9. The system of claim1, wherein the first surface and the second surface are detected fromthe same side of the flow cell.
 10. The system of claim 9, wherein thefirst surface is disposed between an excitation source and the secondsurface during detection by the detection system.
 11. The system ofclaim 10, wherein the first surface and the second surface are excitedby total internal reflection.
 12. The system of claim 9, wherein thefirst surface is disposed between a detector and the second surfaceduring detection by the detection system.
 13. The system of claim 12,comprising corrective optics configured to be inserted and removedbetween the detector and the flow cell after detection of thefluorescent tags on the first surface by the detection system.
 14. Thesystem of claim 13, wherein the corrective optics comprise a lens,objective, or cover slip.
 15. The system of claim 12, wherein the firstsurface is detected through a first objective and the second surface isdetected through a second objective.
 16. The system of claim 1, whereinthe first surface and the second surface are detected from oppositesides of the flow cell.
 17. The system of claim 1, wherein the detectionsystem is configured to produce one or more images of the first surfaceand the second surface.
 18. The system of claim 17, wherein the one ormore images have a resolution of 10 microns or less.
 19. The system ofclaim 1, comprising a radiation source configured to direct excitationradiation toward the first and second surfaces at several differentwavelengths.
 20. The system of claim 19, wherein the detection system isconfigured to capture and detect emitted radiation returned in responseto each wavelength.
 21. The system of claim 1, wherein the detectionsystem is configured to perform confocal detection of fluorescenceemitted from the nucleic acid sites.
 22. The system of claim 1, whereinthe detection system is configured to repeat detection of thefluorescent tags in a process of sequencing the nucleic acids at thenucleic acid sites.
 23. The system of claim 22, wherein the sequencingcomprises sequencing by synthesis.
 24. The system of claim 22, whereinthe sequencing comprises sequencing by ligation.
 25. The system of claim1, wherein the fluorescent reagent comprises fluorescently labelednucleic acids.
 26. The system of claim 1, wherein the fluorescentreagent comprises fluorescently labeled nucleotides.
 27. The system ofclaim 1, wherein each of the nucleic acid sites constitutes a populationof nucleic acids having identical sequences.
 28. A system, comprising: aflow cell comprising first and second surfaces separated by a fluidpassage through which a fluorescent reagent flows to add fluorescenttags to biological sample sites distributed on the first and secondsurfaces; and a detection system configured to detect the fluorescenttags to distinguish the biological sample sites on the first surface,and to detect the fluorescent tags to distinguish the biological samplesites on the second surface.